This invention relates generally to blood diluents for blood cell counting and sizing. More particularly, this invention relates to an improved multi-purpose blood diluent for use in the hematological enumeration of blood cells and the determination of hemoglobin concentration, wherein electronic particle analysis using various scanning devices is obtained. Because of this, the solution must be a stable water solution including chemical salts to provide an electrolytic solution together with other components to obtain certain desirable characteristics.
Routinely, a medical diagnostic procedure includes analyzing and testing blood samples of a patient in order to make certain classic determinations relative to the blood sample. Instrumentation which will accept a patient's blood sample and process the sample automatically and continuously is described in U.S. Pat. Nos. 4,282,902; 4,165,484; 4,240,029 and 4,110,604. The multi-purpose blood diluent described and claimed in this application is suitable for use in such instrumentation as described in these patents, as well as other electronic particle counting instrumentation for analyzing the components of a blood sample.
Thus, in the use of such instrumentation, it is necessary that accurate and successful dilution of a blood sample is made prior to the analysis. The diluent must include proper electrolyte components so as to operate in the system. By the same token, the diluent must also be appropriate for handling the blood cells so as not to effect any change in the blood cells prior to the analysis thereof. For this reason, the blood diluent must be isotonic and osmotically balanced relative to the solutions in the blood cells. The resistance of the blood cells, for example, to lysing for purposes of hemoglobin determinations must not be altered sufficiently to interfere with satisfactory lysing of red blood cells. The solution must have a neutral pH in order to avoid cell swelling or shrinking. It is well known that red blood cell volume is directly related linearly to the reciprocal of the osmolality of the medium in which they are suspended for a period of time up to a few hours. Thus, when such cells are placed in an isotonic medium matching the osmolality of the plasma, they remain unchanged. Placed in an hypotonic medium, water enters the cell membranes and the cells swell. Placed in a hypertonic medium, on the other hand, the cells shrink, because water is drawn out. In extreme cases, swelling is to such a degree that lysis takes place, or shrinking is such that there is very little water in the cells.
All such suspension mediums or diluents contain preservatives, as will be understood, but the preservatives must not alter cell membrane permeability or produce either swelling or shrinking or any negligible change. Surfactants, which are common ingredients in such diluents can also produce similar changes in the cell membrane. It is important, therefore, to select a proper preservative and surfactant component in the diluent, and to select their concentrations so that the swelling of one can offset the shrinking tendency of the other, for example. Alternatively, the tonicity of the diluent may be adjusted to offset cell volume change tendencies produced by a preservative. The net desired effect is neither swelling nor shrinking.
With this invention, such a diluent with the desired balance of properties is provided. The diluent includes, also, sodium and potassium chlorides as electrolytes, and pH buffers, such as monobasic and dibasic phosphates. Ethylene diamine tetraacetic acid is included in the diluent solution to control and chelate divalent cations.
If blood cells are suspended in an isotonic diluent containing a preservative, the cells will generally swell or shrink. The direction of swelling or shrinking may depend upon the preservative used which may be, for example, bacteriocidal, bacteriostatic, fungicidal or fungistatic. While the bacteriocidal preservatives kill bacteria they do not necessarily kill bacterial spores. The bacteriostats on the other hand, inhibit growth of bacteria and bacteriocidal spores. The weaker static action of a bacteriostat is preferred, in accordance herewith, because it is sufficient and may often be obtained by a lower concentration of the preservative. At any rate, it is a significant factor of the invention, as described herein, that the effects of the preservative component of the diluent solution in effecting any change in the blood cells is balanced by the surfactant component. The surfactant component of an isotonic diluent may also effect blood cells to cause cell shrinking or swelling. Some surfactants, for example, may cause lysis of the red blood cells, and a few might even dissolve the red blood cell membranes.
From the foregoing, it is clear that a preservative which changes the red blood cell volume in one direction, and a surfactant which changes the red blood cell volume in the opposite direction can be used together to produce a neutral effect on the cell volume and it is to this particular arrangement in a blood diluent that this invention is particularly directed. Such a combination of preservative-surfactant may include, in accordance with this invention, a variety of preservatives such as, for example, sodium hypochlorite, phenethyl alcohol, benzyl alcohol, etc. The preferred preservative is 2 phenoxyethanol which may be combined with a polyoxyethylene-polyoxypropolyene glycol non-ionic surfactant containing, preferably at least 80% ethylene oxide. Generally, the surfactant may be, for example, ethylene oxide polypropylene glycol condensation products. Such products are available in the market, and one source of such products are designated "Pluronics", products of BASF Wyandotte Corporation, Wyandotte, Mich. 48192. Other products of BASF Wyandotte which may be used are non-ionic surfactants which are structurally tetrafunctional block copolymers consisting of a backbone of ethylenediamine, to which are attached, at four locations, variable links of propylene oxide to which are attached variable links of ethylene oxide. These are entitled "Tetronic" products of BASF.
In considering generally the conditions for preparing the blood diluent solution, in accordance herewith, it may be well to note that satisfactory results have been achieved by a solution containing within the range of between about 6 and 12 grams per liter of sodium chloride as an electrolyte, and preferably 8.795 grams per liter; within the range of between about 0 and 1 grams per liter of potassium chloride as an electrolyte, and preferably 0.28 grams per liter; within the range of between about 0 and 1 grams per liter of potassium dihydrogen phosphate as a pH buffer, and preferably 0.26 grams per liter; within the range of between about 0 and 5 grams per liter disodium hydrogen phosphate as a second pH buffer, and preferably 2.35 grams per liter; within the range of between about 0 and 1 grams per liter disodium ethylenediamine tetraacetic acid, and preferably 0.36 grams per liter; within the range of between about 0.5 and 10 milliliters per liter of a preservative and preferably 2.0 milliliters per liter of a preservative; and within the range of between about 0.001 and 10 grams per liter of a surfactant and preferably 0.5 grams per liter. In this connection, it will be understood that it is one of the features of this invention to control the surfactant, and preservative content of the solution within the ranges noted, so that the effects of one on the mean corpuscular volume (MCV) of the red blood cells (RBC) added will be offset by the effects of the other.
As discussed above, it is important to select the preservative which will provide the desired preservation of the solution and which will inhibit growth of bacteria and bacterial spores without undue action on the blood cells present in the sample under consideration. Any reaction of the preservative, in accordance herewith, on the sample is to be balanced by the nature of the surfactant present. In the selection of the preservative, it is important that the effect on the sample be "mild" in its action. This avoids the need for exceptional accuracy in matching the concentrations of either the preservative or the surfactant reagent or their time courses of action. That is, the use of a slow-acting preservative and a fast-acting surfactant (or vice versa) may result in a neutral condition of no net volume change at final steady-state conditions. However, during the transient condition in order to obtain the stable condition, a mismatch in effects may prevail and cell volumes may change, resulting in erroneous assays. Thus, in the ideal case, neither the preservative nor the surfactant will change cell volumes at all by themselves. Such an ideal is not present, and it is important, therefore, to select the balance between the two so that their concentrations optimize the desirable properties of each.
Thus, the preferred preservative in accordance with the blood diluent of this invention is 2-phenoxyethanol, present in a concentration of 2.0 milliliters per liter. The 2-phenoxyethanol is relatively mild in its reaction to blood cells in a sample under investigation. Moreover, it effectively balances against the results of the surfactant selected in accordance with this invention. One useful surfactant in this invention is PLURONIC F68 which is a non-ionic surfactant of polyoxyethylene-polyoxypropylene glycol containing 80% ethylene oxide. PLURONIC F108 is especially preferred and contains ethylene oxide also in the amount of 80%. It is present in the diluent in a concentration within the range of between about 0.001 and 10 grams per liter, and preferably 0.5 grams per liter. Other PLURONIC as well as TETRONIC surfactants may also be used which vary in their ethylene oxide content. These compounds are described in detail in U.S. Pat. No. 2,674,619 which patent is hereby incorporated by reference in its entirety. That patent describes the preparation of the polyoxyethylene-polyoxypropylene compounds useful as the surfactants in this invention.
With the foregoing and additional objects in view, this invention will now be described in more detail, and other objects and advantages thereof will become apparent from the following description, the accompanying drawing, and the appended claims.